Productive viral replication requires the expression of viral genes in the context of an infected host cell. By studying host-virus interactions involved i viral gene expression, the underlying biology of both the pathogen and the host are uncovered. The Kaposi's sarcoma-associated herpesvirus (KSHV) is a double-stranded nuclear DNA virus and an important human pathogen that causes several lymphoproliferative disorders. The KSHV ORF57 (Mta) protein is conserved among herpesviruses and is essential for viral replication, so elucidation of its mechanisms informs our understanding of the KSHV life cycle. ORF57 has been proposed to function in nearly every stage of mRNA expression from synthesis to processing to translation, but emerging data show that a central function of ORF57 is to increase the stability of viral RNAs in the nucleus. We recently described a cellular nuclear RNA decay pathway that involves the poly (A)-binding protein PABPN1 and poly (A) polymerases PAP?/?, and preliminary studies strongly suggest that ORF57 protects viral RNAs from this decay pathway. The work proposed here seeks to uncover the mechanistic links between KSHV ORF57 and host- mediated nuclear RNA decay.
Specific Aim 1 uses an ORF57-null KSHV BACmid clone to test the importance of ORF57-mediated inhibition of nuclear RNA decay in the context of lytic reactivation. In this aim, we further test whether this activity is conserved amon herpesviruses.
Specific Aim 2 will determine the molecular mechanism by which ORF57 inactivates this cellular RNA decay pathway.
Specific Aim 3 seeks to define the cis- and trans-acting factors that make certain RNAs ORF57-respononsive while other transcripts are relatively unaffected by ORF57. ORF57 is absolutely required for replication, but its mechanisms remain largely unknown, so this work informs KSHV biology by defining the activities of an essential KSHV factor. In addition, PABPN1-mediated decay is a recently described discovered host pathway whose importance is only beginning to be realized, but it appears to be widespread. As such, the proposed studies will significantly impact the current understanding of the molecular mechanisms of gene regulation for both virus and human cells.
This project examines the strategies that viruses use to promote the expression of their genes, which is fundamental for understanding pathogenic viruses. In addition, when viruses infect a human cell, they use the human cell's machinery to promote their own gene expression. Therefore, by determining how viruses regulate their genes, the mechanisms of human gene regulation will also be discovered.